Botulism is a neurological disorder caused by an exotoxin from the organism Clostridum botulinum. The disease is characterized by progressive muscle weakness that can result in complete flaccid paralysis. Unfortunately, there is no known cure for the disorder. Botulinum neurotoxins are classified by the CDC as one of the six highest-risk threat agents for bioterrorism due to their relative ease of production, extreme potency and duration of paralytic activity. Countermeasures are needed to counteract the pathophysiology of BoNTs. To date there are no current interventions that can reverse the effects of intoxication after the toxin has reached its target inside the cell. As such the overarching goal of our proposal is to uncover molecules that can act within an intoxicated cell to provide symptomatic relief to BoNT/A. There are seven serologically distinct serotypes of BoNT, however, we will only focus on BoNT/A as it exhibits the most sustained intoxication and therefore represents the greatest threat of any of the BoNTs. Working within this framework we have taken a two-pronged approach to define such molecules. The first is based on small non-peptidic molecules that can inhibit the intracellular agent that causes neurotoxicity, a protease. For this initiative we will prepare mechanism-based inhibitors as well as team up with ASDI for the high throughput screening of the BoNT/A protease using their diversity collection and custom libraries to both discover new leads as well as enhance the potency of our previous lead compounds with confirmed anti-botulinum properties in vivo. Our second initiative relates to the discovery of molecules that will promote the release of acetylcholine in intoxicated cells. Our goal here are to find an intervention that could repair an intoxicated cell. To accomplish this aim we will take advantage of the known differences in potency based on their nerve terminal mechanism of action between BoNT/A-/E to devise a small molecule plan for neurotransmitter release from an intoxicated. In general we know relatively little about truly effective ways to counter toxin action at the 11th hour, and thus total reliance on any particular intervention is likely to be less than satisfactory. Furthermore, there is a vast difference in the time course of action of current potential antagonist and the toxin. This poses an enormous challenge in terms of discovery of agents for effective antagonism of BoNT/A poisoning. Against this backdrop our research will embrace the discovery of molecules that will provide both immediate and possible long-term relief from these neuroparalytic effects of BoNT/A. RELEVANCE (See instructions): Botulism poses an extreme threat to public health, primarily because of a complete lack of drugs available to combat this disease. Given the bioterrorism threat that botulinum neurotoxin poses, our proposal will develop new molecules to treat botulism and perform the studies needed to advance a potential drug into clinical trials.